The Underwhite Gene, a new Mutation in the Mongolian Gerbil

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Contents

Origins

Over the years it has been the mouse coat colour genes that have played a significant role in the understanding of the basic aspects of mammalian genetics. This holds true for the Underwhite locus which encodes for the MATP (membrane-associated transporter protein) protein, which has been used repeatedly as a coat colour marker in gene linkage and mapping studies. For many years, it was the phenotypic marker of choice for locating genes on chromosome 15. This protein, along with the P protein is known as a melanosome transporter protein and as such share similar qualities. Both have 12 transmembrane regions which are predicted to function as transporters.

These similarities can also be observed in the clinical presentation between patients of OCA 2 (P protein) and OCA 4 (Matp) and is similarly seen in their corresponding homozygous mouse mutations, p/p and uw/uw, where the extent of pigmentation and also the ratio of synthesised eumelanin versus phaeomelanin are very similar. It is variants in both MATP and P genes that account for the natural variations in human pigmentation. The locus has been found to be a key factor in the process of mammalian pigmentation.

The MATP protein is also highly conserved, and especially so in mammals. The human MATP protein is 82% identical to the corresponding mouse MATP protein, 79% identical to the rat MATP protein. It is also 35% identical to the zebra fish MATP protein, 31% identical to the mosquito MATP protein, and 30% identical to the fruitfly MATP protein.

Matp also shares homology with well known plant transporters, especially H+/sucrose symporters. The five amino acids that are conserved in all known plant H+/sucrose symporters are exactly conserved in both the human and mouse Matp. The job of the plant symporters is to couple the transport of a sucrose molecule along with a proton along a proton gradient, and they often facilitate in osmolarity. Even though there are no known sucrose transporters in mammals, the function of Matp maybe to transport a different sugar or similar molecule coupled with proton movement.

Pigment synthesis

In order for a pigment cell to make melanin, tyrosinase must first be correctly targeted to the melanosome, and in turn, its substrate, tyrosine, must then be transported inside the melanosome. On top of this, optimal conditions inside the melanosome that favour enzymatic activity of tyrosinase must also be in place. All of these processes are affected by pH and ionic conditions. Correct Ion transport across the membrane of the melanosome is critical in the biosynthesis of melanin. Two proteins, MATP and the P protein are critical components in the pigment pathway for normal pigmentation to occur, and are likely involved in the ion flux across the membrane of the melanosome.


The Matp gene encodes a protein of -58kDa with twelve membrane-spanning domains that are probably associated with the membrane of the melanosome, and it is thought likely that the protein functions as a transporter. As mentioned above, one of its closest orthologs, encodes a plant sucrose/proton transporter, and thus the Matp may play a role in pH and/or the osmotic regulation of the melanosome. When we look at the melanocytes of both the Matp and P protein mutations tyrosinase is incorrectly processed and targeted. In research, Matp uw cells express normal levels of tyrosinase, and the tyrosine itself is partially delivered to, and is active in the melanosomes. A small portion of this active and correctly processed tyrosinase remains inside the melanocytes and thus contributes to the low levels of melanin that gets produced. This explains the much reduced, but nonetheless significant pigmentation seen in Matp uw mutations. Studies show that the Matpuw melanocytes secrete into the medium dark vesicles that contain tyrosinase, Tyrp 1 (tyrosine related protein 1)and Dct (dopachrome tautomerase) The amount of tyrosinase synthesised in wild-type and Matpuw mutants are roughly similar, however, tyrosinase activity in Matpuw is reduced to around 20% of the level of wild-type melanocytes due to the misrouting of tyrosinase along the secretory pathway.

Along with a reduction in pigment content, it appears that through an electron microscope the melanocytes of Matp mutants appear relatively normal; however, their melanosomes do not. The melanosomes are small and irregularly shaped, and appear as raisin like, crenated structures, containing minimal amounts of melanin. These observations are consistent with a disruption in osmotic regulation.

MATP and the P Protein therefore play a crucial role in the modulation of the processing and intracellular trafficking of tyrosinase and other melanosomal proteins. Matp mutations cause a disruption of this pigment pathway, the net result being the abnormal release of tyrosinase and Tyrp 1 into the medium. Therefore, because tyrosinase is the key rate-limiting enzyme of melanogenesis, it results in the phenotypes we see in Matp mutations. The similarities of the two proteins, both being predicted as transport proteins involved in the trafficking of melanosomal proteins, provides science with vital clues as to the roles of such proteins in regulating mammalian pigmentation.

The mouse Matp gene and its mutant alleles

Mutations in the mouse Matp gene have given rise to an unusual series of alleles known as underwhite that display a varied spectrum of pigment phenotypes. The effects of the mutation are restricted to the melanocytes, and all the variants of the existing mutations are associated with small, crenated melanosomes that range in their amounts of pigment that they hold.

The original underwhite mutant allele in mice arose spontaneously in a population of C57BL/6J strain of lab mice. The gene is inherited as a simple Mendelian recessive, and was first described by M.M. Dickie in 1964. Subsequently, two other recessive alleles have also been described, these being underwhite intense uw(i) and underwhite dense uw(d) (Sweet et al. 1998) Underwhite dense arose spontaneously in a population of TF/Le strain of lab mice. Underwhite intense appeared in a recovery of frozen C57BL/6J underwhite frozen embryos in 1994. Crosses to other mice carrying the already known alleles of underwhite suggested that it was either a new mutation of underwhite or a mutation in a modifying gene. However, due to poor reproduction this line was lost before further genetic tests were completed. A fourth allele, this time a semi-dominant allele to underwhite, was also described in the literature (Mackpike and Mobraaten 1984) The allele behaves in a semi-dominant fashion over the wild-type allele for full colour and to all the other recessive alleles on the underwhite locus. Named as underwhite dominant brown Uw (dbr)  ,this mutation arose in a population of B10.PL (73NS)/Sn congenic lab mice which was transferred by backcrossing to the C57BL/6J background.

Dominance heirarchy of the mutant alleles in mice

Uw (dbr) - Underwhite dominant brown
Uw- Full colour (wild type allele)
uw(d) –Underwhite dense
uw –Underwhite

Coat and eye colours of the mouse mutants

Underwhite uw

On homozygous uw/uw mice the coat colour is a light buff/beige with white underfur. The eyes are pink at birth but darken to a dark ruby with age. On either agouti or non-agouti background the coat colour and eye colours remain very similar.

Underwhite Dense uw(d)

Mice homozygous for underwhite dense uwd/uwd have a much more pigmented coat than homozygous underwhite mutants , which are classed as having the most severe phenotype. On a non-agouti background the mice are a grey/brown colour. The eyes are a very dark ruby which to the untrained eye appears black. The ventrum is slightly lighter coloured than the dorsum and also the ears are lighter. On an Agouti background the banding pattern is almost identical to agouti chinchilla (A/A Tyr-c-ch/Tyrc-ch)mice. Low amounts of tyrosinase activity results in the chinchilla agouti mouse having hairs that are banded grey and white, therefore the almost identical phenotype seen in uwd/uwd may indicate that uw deficits also serve to reduce tyrosinase activity.

Underwhite Dominant Brown Uw (dbr)

Homozygous underwhite dominant brown Uwdbr/Uwdbr mice have a light beige coat (only slightly darker than underwhite) The eyes are pink at birth but darken with age. Heterozygous mice for this mutation Uwdbr/+ have dark brown coats.

The human Matp gene and OCA4

Oculocutaneous albinism (OCA) affects around 1/20,000 people worldwide. Reduced pigmentation as the eye is developing can result in misrouting of the optic nerves, nystagmus, alternating strabismus, and reduced visual acuity. A lack of pigment in the skin leaves it vulnerable, which, in turn, leads to an increased risk of skin cancer.

OCA1- Involves mutations of the TYR gene encoding tyrosinase. As mentioned earlier, this is the rate-limiting enzyme of the production of melanin pigment. This form of OCA accounts for around 40% of OCA cases worldwide.

OCA2- Regarded as the most common form of OCA and accounts for around 50% of OCA cases worldwide. It is associated with mutations of the P gene

OCA3- Known as red/rufous albinism, and considered a rare form of OCA. It is associated with mutations in Tyrp1, encoding tyrosine related protein 1.

OCA4- Is a recently identified form of OCA and involves mutations in the MATP gene. This form was based on the underwhite mouse phenotype as a candidate for Matp as a form of OCA. Its phenotype is similar to OCA2. While this form of OCA is rare worldwide, it is more common in the Japanese population. There have also been case accounts in  German, Korean and Turkish populations.

Mutations of the Matp gene in other species

Apart from the MATP gene sharing significant homology with fruit flies and genes as distant as plant proton/sucrose symporters, mutations in the MATP gene also cover several species. Most notable is probably the Cream locus (Ccr) in horses which has been identified as a mutation of the MATP locus. The gene mutation that gives rise to the cream colour in horses is associated with Matp uwdbr (underwhite dominant brown). There exists similar mutations in other species which all produce a hypopigmented phenotype. Polymorphs in MATP exist in Medaka fish, chimpanzees, Norway rats, Japanese Quail, dogs, and cattle (Bos Taurus)

The Underwhite Locus in the Mongolian gerbil: A hypothesis

The Grey Agouti, and indeed the entire G locus has always remained a relative mystery in gerbil genetics. We could visibly see how the mutant gene affected the coat colour; it diluted eumelanin and removed most of the phaeomelanin pigments, but at the molecular level things remained shrouded in mystery.

When researching the grey gene, breeding tests were conducted to see if the gene was allied to the C locus. This approach was logical as the Grey agouti closely resembled the Agouti chinchilla mouse, and not only this, but the chinchilla mutation is highly conserved in many domestic species and was expected to appear in gerbils. Allelic breeding tests proved this wasn’t the case, and to this date, neither chinchilla (cch) nor albino (cc) have appeared in the gerbil despite both being common mutations in other species.

When comparing the grey mutant phenotype to other grey mutations in other species they had relatively few known examples to choose from. These were grey-lethal and grizzled in the house mouse and dark grey and lethal grey in the Syrian hamster. However, both grey-lethal and lethal grey are known to cause the demise of the homozygotes, while grizzled and dark gray animals tend to be undersized and have reduced viability. This wasn’t the case for the Grey mutation that had appeared in Mongolian gerbils, which were of a normal size and had no fertility problems whatsoever. With nothing else to go on, the mutation remained unique and was tentatively allocated a locus of it own, namely the G locus (Grey)

With the appearance of the cream mutant it helped shed some light on the G locus. Here was a recessive gene, that when homozygous on ‘A-’ or ‘aa’ backgrounds will produce a cream coloured gerbil with dark ruby eyes. Through breeding trials it was shown to be associated with alleles of the G locus and this in turn helped identify the possible locus of this particular set of unusual mutant alleles. Based on breeding tests, homologous behaviour of gene action, and phenotype comparisons with the house mouse, the closest candidate in our opinion are the mutant alleles of the underwhite locus.

The Cream Mutant

(synonyms: g(x), satin)

The homozygous cream gerbil gives us a deep cream fur with dark ruby eyes, on either an A- or aa backround which has the potential to be a much superior cream than present gene combinations which give us the white bellied creams (Ivory & Apricot) or self cream (c-separator, red-eyed silver nutmeg). Because the mutation is restricted just to the melanocytes (it only affects pigment and doesn’t have adverse related effects) it is a very healthy mutation as well.
The mutation resembles the underwhite mutant of the house mouse, and more closely still, the underwhite intense mutation in the underwhite mouse series of alleles.

As you can see from the underwhite and underwhite intense mice in the photo, the mutated gene removes most of the pigment, leaving a little phaeomelanin left in the coat hairs. This is similar to what is happening in the cream mutation of the gerbil, and the way the gene behaves in that fact that the ratio of phaomelanin to eumelanin resembles (although more severe) the action of the pink-eyed dilute mutation, also holds true for this corresponding mutant in the gerbil. The mice mutants are born with pink eyes but these get darker as they reach maturity, and this is also how the gene behaves in the gerbil; gerbil pups are born with pink eyes which darken as they age.  If we look closer at the underwhite series of alleles we can see close similarities between our well known Grey mutation and the mouse mutation underwhite dense.

 

 

The Grey Mutant

Grey Agouti



 

The grey mutation in gerbils very closely resembles the phenotype of the underwhite dense mouse. On a non-agouti background it produces the well known slate coat colour. The gerbil has very dark eyes, almost black that shine red under a strong light. Its belly is also slightly lighter than its top coat, as are their ears. This phenotype is virtually identical to the underwhite dense mouse, with its dark brown/slate coat, their ventrum being slightly lighter than the dorsum, lighter ears,and with deep ruby eyes that look black to the untrained observer.

Being much harder to ascertain (as most studies for underwhite were being carried out on a non-agouti background) was the phenotype of the underwhite dense mouse on an agouti background. However the latest research on this mutation points to it mimicking the hair banding pattern of the Agouti chinchilla mouse. This current data confirms that the Grey mutant in gerbils most closely resembles this phenotype in the mouse.


When we also compare underwhite dense in breeding tests we can see comparable phenotypes in test crosses. In the paper, The Underwhite(uw) Locus Acts Autonomously and Reduces the Production of Melanin –Anne L. Lehman, Willys K. Silvers, Neelu Puri, Kazumasa Wakamatsu, Shosuke Ito, and Murray H. Brilliant, the researchers produced double mutant phenotypes consisting of combinations of uw(d) with pp and ee. The double mutant combining uw(d)uw(d) to ee was produced on a non-agouti background and the double mutant, uw(d)uw(d)pp was produced on an Agouti background. The resulting phenotypes of these double mutant offspring are comparable to what we would see in gerbils as you can see in the photo on the left;


You have to bear in mind that the pink-eyed dilute mouse mutation has a stronger effect on the mouse coat pigments than the comparable pink-eyed dilute mutation in gerbils. You can see this visually demonstrated if we compare the ‘aapp’ Lilac gerbil coat colour to the ‘aapp’ Dove coat in the mouse. In the mouse the double mutant phenotype of uw(d)uw(d)pp it has been produced on an Agouti background by the researchers and gives rise to a pink-eyed white phenotype. In gerbils the similar genotype (A-ggpp) gives us our white-bellied cream phenotype, however this has a tendency to fade with age and often resembles a ruby-eyed white.

The same holds true for the ‘aaee’ mouse. Here we have a yellow mouse because the effect of ‘e’ completely masks the black. In gerbils ‘e’ can only partially mask black and the result is the nutmeg phenotype. The double mutant offspring from crossing uw(d) to ee on non-agouti mice results in a light cream coloured phenotype. With only a partial masking of black this coat colour would be very similar to our silver nutmeg (aaeegg).

Hidden clues to a Cream locus

In other species such as the horse, this particular locus is regarded as a Cream locus, here though the gene responsible is Uw (dbr). However if we had taken a closer look at our old “Grey” locus, we can still see how the “grey” mutant and also uw(d) is intrinsic to the production of cream coat colour variants, and also how all the known alleles on this locus are used to create cream coat varieties.
 
When we start to examine the action of the uw(d) allele on pigment production, and compare this to uw and Uw(dbr) we can begin to see how closely all three alleles behave on the yellow pigments in the fur. uw(d) behaves like an intermediate allele, has a preference for removing yellow pigments in the coat, and marginally dilutes the black pigments.  However, it can't remove all the yellow pigments in the fur and this is why many a Grey Agouti can still have some yellow pigments remaining in their coat.  All the mutant alleles on this locus, be it uw, uw(d) or Uw(dbr) act in exactly the same manner where the yellow pigments are concerned, and these pigments are diluted from an intense yellow to a cream colouring.  The main difference in these three mutant alleles on pigment production is how they behave on the black pigments.  In this situation, uw and Uw(dbr) more or less stop the production of the black pigments, where as with the uw(d) allele it is only able to dilute the black pigments.
 


 

Click for Larger Image So knowing these similarities between the alleles regarding their dilution of the yellow pigments to produce cream colours, we can then begin to test just how apparent this is with uw(d) too if we then take the black pigments out of the  equation.  An effective way to do this is to use the pink-eyed dilution gene. Pink-Eyed dilution will strip most of the black in a gerbils coat to give us three well known red-eyed, yellow coated varieties; these being  the Argente Golden, Red-Eyed Honey and Saffron. Now if we look at the uw(d) variants of these three coat colours we can see at a glance how they all produce cream fur, these being Ivory, Apricot and the Self cream coat colour varieties.
 
 
 
Cream variants with recessive yellow
 
We can see similar things happening if we use recessive yellow in uw(d) crosses, however molecular studies show that although both the mouse and gerbil mutations reside on the Extension locus, the form of recessive yellow in the gerbil is a quite dissimilar mutation to recessive yellow in the mouse, and as such behaves quite differently. In the mouse, recessive yellow produces yellow furred pups that range in “sootiness”, but as they age and go through successive moults, the sootiness disappears leaving the adult with a yellow coat. In the gerbil we have the opposite happening, and pups, whether A- or aa are born with yellow fur, however as they age and go through successive moults, the black pigments on the coat begin to return leaving us with colours that are unique on both Agouti and non-agouti coats.
 
So when we look at the uw(d) versions of recessive yellow on both an Agouti and non-agouti background, both A- and aa fur on the pups and juveniles are changed from their usual  yellow to a cream colour, but as they age the familiar black pigments begin to return with each moult and start to transform the coat into its adult colouring
 
Click for larger Image

 The return of the black pigments in the adult and the subsequent dilution of it by uw(d) gives rise to two rather unique coat colours on both Agouti and non-agouti. The  uw(d) version of recessive yellow on an Agouti background  becomes a cream variant, and like all other cream variants it will fade as it ages to an Ivory shade, but also, because some of the black pigments return as an adult, the normal adult black “coal-dust” sprinkling that appears over and around the dorsal area and face regions, then appear to be diluted to grey in the uw(d) variant.
 
 
 
Click for larger ImageOn a non-agouti  background  the effect is very dramatic once adulthood is reached and the cream coloured coat of the juvenile fades to an Ivory colour as it ages, but with each successive moult the gene allows more black pigments to return into the fur until it overwhelms the faint cream colouring that remains. The end result is akin to a much darker, self version of the Grey Agouti.
 
 
 
 
 
Click for Larger ImageIn the research paper above (Lehman, Silvers, et al.) it gives us an interesting glimpse
at what the uw(d) variant of the recessive yellow mouse looks like on a non-agouti background, and also what our Silver Nutmeg may look like if recessive yellow in the Mongolian gerbil were more similar to its mouse counterpart.  With recessive yellow in the mouse the black pigments remain absent into adulthood and we are left with a very striking Black-Eyed Cream mouse.  When taking all these facts into consideration, we believe we can associate the phenotypes of both the cream and the grey mutant in the Mongolian gerbil to the underwhite locus and its corresponding alleles in the mouse.

 

Ed Cope

References

Mutations in the Human Orthologue of the Mouse underwhite Gene (uw) Underlie a New Form of Oculocutaneous Albinism, OCA4- J. M. Newton,1Orit Cohen-Barak, Nobuko Hagiwara, John M. Gardner, Muriel T. Davisson, Richard A. King, and Murray H. Brilliant.

A mutation in the MATP gene causes the cream coat colour in the horse-Denis MARIAT, Sead TAOURIT, Gérard GUÉRIN

A New Allelic Series for the Underwhite Gene on Mouse Chromosome 15– H.O. Sweet, M.H. Brilliant, S.A. Cook, K.R. Johnson, and M.T. Davisson

The Underwhite(uw) Locus Acts Autonomously and Reduces the Production of Melanin–Anne L. Lehman, Willys K. Silvers, Neelu Puri, Kazumasa Wakamatsu, Shosuke Ito, and Murray H. Brilliant.

Tyrosinase processing and intracellular trafficking is disrupted in mouse primary melanocytes carrying the underwhite (uw) mutation. A model for oculocutaneous albinism (OCA) type 4- Gertrude-E. Costin, Julio C. Valencia, Wilfred D. Vieira, M. Lynn Lamoreux and Vincent J. Hearing.

Identification of Aim-1 as the underwhite Mouse Mutant and Its Transcriptional Regulation by MITF- Jinyan Du and David E. Fisher.

Gray mutant in the Mongolian gerbil- Leiper, B.D. & Robinson, R. 1985-The Journal of Heredity, 76, 473.

The pigmentary system-James J. Nordlund, Raymond E. Boissy, Vincent J. Hearing, William Oetting, Richard A. King, Jean-Paul Ortonne